Metabolic reconstruction of H. influenzae

A commenter on Wednesday's post suggested that I consider using the available 'metabolic reconstruction' of H. influenzae to assess whether the nucleotides from DNA uptake would make a significant contribution to growth. "Metabolic reconstruction?", I said to myself. Sounds like something I ought to know about.

So I found the papers. The first one appeared way back in 1999, but I've been reading a slightly more recent one (Schilling and Pallson, 2000. Assessment of the metabolic capabilities of Haemophilus influenzae Rd through a genome-scale pathway analysis. J. Theor. Biol. 203:249-283). Metabolic reconstruction uses computer simulations of the catalytic activities of the proteins encoded in a genome to infer as much as possible about the organism's metabolism.

H. influenzae was done first only because its genome sequence was the first to be available. I suspect that more recent analyses of better-known organisms are more sophisticated, but I don't expect anyone to go back and redo the H. influenzae analysis (though maybe that's too pessimistic).

What Schilling and Palssen did: They included only proteins that are metabolic enzymes or membrane transporters - this is less than 25% of the proteins encoded by the genome (~400 genes, 461 reactions). This includes some reactions for which they had biochemical evidence but not identified genes. Their model metabolism did not include any effects of regulation of gene expression or (I think) of regulation of protein activity. No transcription or translation or DNA replication. The model included 51 metabolites required for biomass generation and maintenance (amino acids for protein synthesis, NTPs and dNTPs for RNA and DNA synthesis, phospholipids for membranes, etc).

They simplified the model metabolic structure by subdividing it into six subsystems: amino acids (A), nucleotides (N), vitamins and cofactors (V), lipids (L), central metabolism (C) and transport/energy/redox (T). For each of these they then identified the 'extreme' pathways that (I think) set the metabolic limits. Finally, they used their model to investigate H. influenzae's metabolic capabilities.

Their conclusions are interesting, though some of them are clearly wrong (i.e. their model predicts X, but we know X is not true for real H. influenzae). But the reason I'm reading this paper is to find out whether this model can be used to make predictions about the effect of DNA uptake on metabolism and on growth. I was thinking that the answer is No, mainly because of the simplifying assumptions the model had to make.

The fundamental problem is that I would like a quantitative answer, such as "Taking up 200 kb of DNA per generation would increase the growth yield by 0.5%". But I think that the model will only give qualitative answers about capabilities, such as "Yes, taking up DNA could increase the growth rate". I suppose that kind of answer might be useful, enabling us to make the important distinction between it and "No, taking up DNA can't increase the growth rate, because reaction A is missing".

How would getting one or the other answer change our thinking? If we got a "No, because..." answer, the first step would be to track down the reason(s) and find out whether we'd expect them to hold true in real cells. Is the needed gene really missing? Do we have other evidence that this reason will not apply? (For example, the model predicts that H. influenzae would not be able to use citrulline as its pyrimidine source because a gene is missing, but real cells can do this and we have identified the supposedly-missing gene.) If the reasons for the "No" answer did hold up under scrutiny, we'd need to seriously rethink our hypothesis that cells take up DNA as a nutrient source.

What if we got a "Yes" answer? I don't think we'd do anything differently than we're doing already, but we might be slightly more confident that our hypothesis is reasonable.

I think a genuine "No" answer is unlikely in principle, because, to use the DNA they have taken up for new DNA synthesis, all cells need to do is rephosphorylate the dNMPs that result from nuclease degradation of this DNA. Do H. influenzae have the pathway to do this? They do have enzymes that can catalyze these reactions, but I don't know enough biochemistry to be absolutely certain that these reactions will proceed efficiently.

Could cells use the dNMPs as sources of NTPs for RNA synthesis? This is more complex, as the deoxyribose sugar needs to be removed from each base and replaced with a ribose sugar (cells have no enzyme that could simply create ribose from deoxyribose by adding back the missing oxygen). What cells actually do is remove the deoxyribose and phosphate from the dNMP, and then add ribose plus phosphate to create a NMP which can then be phosphorylated into a NTP. These reactions are included in the metabolic reconstruction; they consume an ATP, but much more will be saved by not having to synthesize the NMP from scratch. The cells also get some energy by metabolizing the deoxyribose-1-phosphate this releases, although this isn't included in the reconstruction. I just checked the pathway: the deoC gene product converts deoxyribose-1-phosphate into glyceraldehyle-3-phosphate plus acetaldehyde - the former feeds into glycolysis but I don't know what happens to the acetaldehyde (maybe ADH converts it to alcohol?).

What about using the dNMPs as sources of carbon (= energy), nitrogen and/or phosphate? Presumably this would only happen when the cell had satisfied its need for dNTPs and NTPs for DNA and RNA synthesis. Our visitor told us that at least some bacteria lack the enzymes needed to break down purine and/or pyrimidine bases; such a cell couldn't use nucleotides as nitrogen sources, and using the sugar for energy might leave cells clogged up with the leftover bases.

This probably isn't a big issue for H. influenzae, as I expect it to only use dNMPs for DNA and maybe RNA synthesis. But if I'm hoping to show that adding DNA to a B. subtilis culture improves growth yield, knowing whether or not it can use the bases could make a big different to my expectations. I've found a paper (Schultz et al. J. Bact. 183:3293) showing that B. subtilis can metabolize purine bases to NH3 and CO2 (with no energy gained but none expended). And an earlier paper from the same group (Per Nygaard's) gives an overview of nucleoside catabolism by B. subtilis; the basic conclusion is yes they can.

So it looks like feeding B. subtilis DNA is a reasonable idea.

2 comments:

I suggest you doing the real experiments several months later. Some controls lacked in Finkel's experiments. What's more, The only evidence just comes from the gene inactivation experiment and again some necessary controls are not included. Complementary experiments (express corresponding competent genes in-trans in the mutants) have not mentioned in these experiments. Before expanding the concept that DNA can be used as a nutrient, it is better to firmly confirm this in E. coli.

"expanding the concept that DNA can be used as a nutrient"? It hardly originated with E. coli - we first published it in 1993, and Steve Finkel got the idea from one of my students. (Sorry to bitch; it's a sensitive point.)

E. coli is not a very good system for this, as nobody has yet identified conditions where cells take up measurable amounts of DNA. A naturally competent autotroph like B. subtilis is a much better bet.